Prolylendopeptidase was first found in human uterus by Walter et al., in 1971 as a specific endopeptidase which cleaves a peptide at the carboxyl- terminus side of a proline residue (Walter, R. et al. Science, 1971, 173, 827-829), and ever since the enzyme has been continuously studied with regard to its physiological role.
On the other hand an endopeptidase showing a very similar specificity to mammalian prolylendopeptidase was found from a bacterium, Flavobacterium meningosepticum in 1978 (Yoshimoto, T., et al. Agric. Biol. Chem. 1978,42,2417-2419). This finding enabled a larger amount to be prepared (but still at a lab scale) of the enzyme, and prolylendopeptidase became available for specific cleavage (Yoshimoto, T., et al. J. Biol. Chem. 1980, 255,4786-4792.) of proteins and peptides. Its unique specificity, recognizing proline residues, makes the enzyme quite useful as a basic tool of protein engineering and draws more attention to the study of the structure and function relationship. The preparation of prolylendopeptidase from F. meningosepticum, however, has the following two crucial drawbacks arising from the bacterium. 1) The bacterium is pathogenic (Yoshimoto, T. et al., 1978, supra; Buchanan, R. E. et al. "Bergey's Manual of Determinative Bacteriology," 8th ed. 1974, The Williams & Wilkins Co., Baltimore.) and 2) it produces not only prolylendopeptidase but also significant amounts of other specific or non-specific peptidases (Yoshimoto, T. et al., 1978 supra). These problems have prevented industrial production of the endopeptidase, in spite of a growing demand for the enzyme.
Japanese Unexamined Patent Publication (KOKAI) No. H2-5880 describes cloning of post-proline peptidase gene derived from Bacteroides gingivalis, but this enzyme is clearly different from the present enzyme in that the former cleaves glycyl-proline-4-methoxy-.beta.-naphtylamide which cannot be cleaved by the present prolylendopeptidase.
D. Rennex et al., Biochemistry 30,2195-2203, 1991 describe a cloning of cDNA for prolylendopeptidase from the porcine brain, but does not describe an expression of the cDNA.
It is believed that prolylendopeptidase is useful for modification of peptides, for example, C-terminal amidation of biologically active peptides such as LH-RH, oxytocin, calcitonins or the like, but for this purpose, it is necessary to obtain a large amount of the enzyme. Moreover, the enzyme preparation should be free of other peptidases, to ensure a desired reaction. Thus, the production of the enzyme by a gene recombination process is essential.